Hypocrealean fungi associated with populations of <Emphasis Type="Italic">Ips typographus</Emphasis> in West Carpathians and selection of local <Emphasis Type="Italic">Beauveria</Emphasis> strains for effective bark beetle control

In Slovakia, a diversity of entomopathogenic fungi (Ascomycota, Hypocreales) associated with outbreaks of Ips typographus was studied in 81 localities and as many as 113 in vitro cultures of five entomopathogenic species were isolated from infected individuals: Beauveria bassiana (87 isolates), B. pseudobassiana (14 isolates), B. caledonica (6 isolates), Lecanicillium lecanii (4 isolates) and Isaria farinosa (2 isolates). B. pseudobassiana is recorded in natural populations of I. typographus for the first time. Biological properties of selected Beauveria isolates, including colony growth, biomass production, conidia yield and pathogenicity to I. typographus adults, were studied in a series of laboratory bioassays and much intra- and interspecific variability was detected. B. bassiana isolates produced biomass or conidia at significantly higher rate than B. pseudobassiana and B. caledonica isolates. Two B. bassiana isolates were selected as the most virulent to bark beetle adults, demonstrating a mean LC₅₀ ranging from 0.72 to 2.05?×?10⁶ conidia ml^?1, and were qualified as promising candidates for biocontrol of I. typographus. Their virulence was significantly higher than that of the mycoinsecticides Boverol®, which was used as a reference strain in the virulence bioassays.     

Grapevine leaf tissue colonization by the fungal entomopathogen <Emphasis Type="Italic">Beauveria bassiana</Emphasis><Emphasis Type="Italic">s.l.</Emphasis> and its effect against downy mildew

A study was conducted to examine whether Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) can colonize grapevine leaf tissues and subsequently confer protection against downy mildew caused by Plasmopara viticola (Berk. and Curt.) Berl. and de Toni. Following the foliar inoculation of plants with conidial suspensions of selected B. bassiana strains, colonization of leaves by the fungus was determined using culture-based and PCR techniques at different time intervals. Seven days following B. bassiana inoculation, grapevine plants were challenged with P. viticola and symptoms were assessed by calculating the disease incidence and severity. Although all tested strains were able to colonize grapevine plants, percent colonization differed significantly among strains. Disease incidence and severity were, on the other hand, significantly reduced in B. bassiana-inoculated plants compared to control plants irrespective of strain. This study is one of very few studies investigating the promising role B. bassiana could play as a plant disease antagonist.     

The production and uses of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> as a microbial insecticide

Among invertebrate fungal pathogens, Beauveria bassiana has assumed a key role in management of numerous arthropod agricultural, veterinary and forestry pests. Beauveria is typically deployed in one or more inundative applications of large numbers of aerial conidia in dry or liquid formulations, in a chemical paradigm. Mass production is mainly practiced by solid-state fermentation to yield hydrophobic aerial conidia, which remain the principal active ingredient of mycoinsecticides. More robust and cost-effective fermentation and formulation downstream platforms are imperative for its overall commercialization by industry. Hence, where economics allow, submerged liquid fermentation provides alternative method to produce effective and stable propagules that can be easily formulated as dry stable preparations. Formulation also continues to be a bottleneck in the development of stable and effective commercial Beauveria-mycoinsecticides in many countries, although good commercial formulations do exist. Future research on improving fermentation and formulation technologies coupled with the selection of multi-stress tolerant and virulent strains is needed to catalyze the widespread acceptance and usefulness of this fungus as a cost-effective mycoinsecticide. The role of Beauveria as one tool among many in integrated pest management, rather than a stand-alone management approach, needs to be better developed across the range of crop systems. Here, we provide an overview of mass-production and formulation strategies, updated list of registered commercial products, major biocontrol programs and ecological aspects affecting the use of Beauveria as a mycoinsecticide.     

Identification of calmodulin binding proteins in the entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

Calmodulin (CaM) is a primary Ca²⁺ receptor and plays a pivotal role in a variety of cellular responses in eukaryotes. Even though a large number of CaM-binding proteins are well known in yeast, plants, and animals, little is known regarding CaM-targeted proteins in filamentous fungi. To identify CaM-binding proteins in filamentous fungi, we used a proteomics method coupled with co-immunoprecipitation (CoIP) and MALDI-TOF/TOF mass spectrometry (MS) in Beauveria bassiana. Through this method, we identified ten CaM-binding proteins in B. bassiana. One of the CaM-targeted proteins was the heat shock protein 70 (BbHSP70) in B. bassiana. Our biochemical study showed that ATP inhibits the molecular interaction between BbHSP70 and CaM, suggesting a regulatory mechanism between CaM and ATP for regulating BbHSP70.     

Susceptibility of <Emphasis Type="Italic">Diaphorina citri</Emphasis> (Hemiptera: Liviidae) and Its Parasitoid <Emphasis Type="Italic">Tamarixia radiata</Emphasis> (Hymenoptera: Eulophidae) to Entomopathogenic Fungi under Laboratory Conditions

Diaphorina citri (Kuwayama) is a global pest of citrus that transmits the bacteria associated with the disease, Huanglongbing. Entomopathogenic fungi and the parasitoid Tamarixia radiata (Waterston) are important biological control agents of this pest and likely to interact in D. citri populations. As a basis for interaction studies, we determined the susceptibility of nymphs and adults of D. citri and adults of the parasitoid T. radiata to six fungal isolates from the species Beauveria bassiana s.l. (Bals.-Criv.) Vuill. (isolates B1 and B3), Metarhizium anisopliae s.s. (Metsch.) (Ma129 and Ma65) and Isaria fumosorosea Wize (I2 and Pae). We conducted experiments evaluating infection levels in all three insect groups following inoculation with a series of conidial concentrations (1 × 10⁴–1 × 10⁸ conidia mL^?1). Results showed that D. citri nymphs and T. radiata were more susceptible to fungal isolates than D. citri adults. Overall, B. bassiana and M. anisopliae isolates caused the greatest infection compared with I. fumosorosea isolates in all three groups of insects. Isolates B1 (B. bassiana) and Ma129 (M. anisopliae) infected a greater proportion of adults and nymphs of D. citri, respectively. Both isolates of B. bassiana caused greater infection in T. radiata compared with isolates of the other fungal species. We propose that isolates B1 and Ma129 are the strongest candidates for control of D. citri. Our results represent the first report of entomopathogenic fungi infecting T. radiata, and the basis for future studies to design a biological control programme that uses both agents more efficiently against D. citri populations.     

The endosymbiotic bacterium <Emphasis Type="Italic">Wolbachia</Emphasis> enhances the nonspecific resistance to insect pathogens and alters behavior of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

To determine biologically important effects of the cytoplasmic endosymbiont Wolbachia, two substrains of the same Drosophila melanogaster strain have been studied, one of them infected with Wolbachia and the other treated with tetracycline to eliminate the bacterium. Females of D. melanogaster infected with Wolbachia are more resistant to the fungus Blauveria bassiana (an insect pathogen) than uninfected females; infected females also exhibited changes in oviposition substrate preference. Males infected with the bacterium are more competitive than uninfected males. The possible role of Wolbachia in the formation of alternative ecological strategies of D. melanogaster is discussed.     

Dynamics of mortality of the migratory locust under synchronous infection with entomopathogenic fungi (<Emphasis Type="Italic">Beauveria bassiana,Metarhizium anisopliae</Emphasis>) and bacteria <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

As a result of search for species and strains of entomopathogenic fungi and bacteria virulent to migratory locust (Locusta migratoria migratoria L.), combinations were found which cause high mortality of insect in a short time interval. Four or five days after the L. migratoria had been infected with Beauveria bassiana (Bals.) Vuill and Metarhizium anisopliae (Metsch.) Sorokin a sharp increase in nymphas’ mortality was observed, reaching 95–100% on the 13th to 17th day after inoculation. The mortality of L. migratoria after infection with Pseudomonas sp. bacteria was approximately 30–50% on the 3rd to 7th day of the experiment. Later deaths of the locusts were not observed. When we made synchronous inoculation with fungi and bacteria, the rate of nymphas’ mortality was higher in comparison with monoinfections, and LT₅₀ was about three days. Microbiological analysis of the dead insects showed that both pathogens could coexist in the locust. To determine the antagonism between Pseudomonas and fungi on a synthetic nutrient medium, the blocking method was used. We showed that the fungi do not affect the development of the bacterium, and the Pseudomonas has an insignificant effect on the fungi growth.     

Horizontal transfer of the C-termini of <Emphasis Type="Italic">tccC</Emphasis> genes in <Emphasis Type="Italic">Photorhabdus</Emphasis> and <Emphasis Type="Italic">Xenorhabdus</Emphasis>

The high molecular weight insecticidal toxin complexes (Tcs), including four toxin-complex loci (tca, tcb, tcc and tcd), were first identified in Photorhabdus luminescens W14. Each member of tca, tcb or tcc is required for oral toxicity of Tcs. However, the sequence sources of the C-termini of tccC3, tccC4, tccC6 and tccC7 are unknown. Here, we performed a whole genome survey to identify the orthologs of Tc genes, and found 165 such genes in 14 bacterial genomes, including 40 genes homologous to tccC1-7 in P. luminescens TT01. The sequence sources of the C-termini of tccC2-6 were determined by sequence analysis. Further phylogenetic investigations suggested that the C-termini of 6 tccC genes experienced horizontal gene transfer events.     

<Emphasis Type="Italic">Fusarium verticillioides</Emphasis> and fumonisin contamination in <Emphasis Type="Italic">Bt</Emphasis> and non-<Emphasis Type="Italic">Bt</Emphasis> maize cultivated in Brazil

Fusarium verticillioides is one of the main pathogens of maize, causing ear and stalk rots. This fungus is also able to produce high levels of fumonisins, which have been linked to various illnesses in humans and animals. Previous studies have shown that maize hybrids genetically modified with the cry genes from the bacterium Bacillus thuringiensis (Bt) presented lower incidence of F. verticillioides and fumonisin levels, presumably through the reduction of insects, which could act as vectors of fungi. The aim of this study was to assess the incidence of F. verticillioides and the concentration of fumonisins in Bt and isogenic non-Bt hybrids (2B710Hx, 30F35YG, 2B710, and 30F35, respectively). The samples of 2B710Hx and 30F35YG presented lower F. verticillioides frequency than 2B710 and 30F35 samples. However, there was no statistical difference between fumonisin contamination when Bt and non-Bt samples were compared (P > 0.05). The results suggest that other environmental parameters could possibly trigger fumonisin production during plant development in the field; consequently, other management strategies should be applied to aid controlling fumonisin contamination in maize.     

Impact of sequential exposure of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and imidacloprid against susceptible and resistant strains of <Emphasis Type="Italic">Musca domestica</Emphasis>

Insecticide resistance in the housefly Musca domestica is hampering pest management. However, entomopathogens, possibly in combination with insecticides, may have control potential against resistant houseflies. This study investigates the combination of the entomopathogenic fungus Beauveria bassiana and the neonicotinoid insecticide, imidacloprid against a susceptible and a resistant housefly strain, respectively under laboratory conditions. The fungus and insecticide were tested alone and in combinations at LC₃₀. Significant and synergistic interactions between B. bassiana and imidacloprid were observed with increased mortality rates of the combined treatment as compared to individual treatment in housefly strains 772a (susceptible) and 766b (resistant). Significant differences in the GST and P450 activities for both strains were found. Female 766b flies caused 15- to 237-fold increases in gene expression of xenobiotic response genes for B. bassiana and 23- to 120-fold changes for imidacloprid. The combination of B. bassiana and imidacloprid caused significant synergistic interaction when applied against two housefly strains irrespective of order of application. The effect was highest when the insecticide was applied first. The resistant housefly strain had elevated detoxification enzymes and higher expression of detoxification genes, but showed the same level of susceptibility to the combined fungus/insecticide treatment as the susceptible strain.     

<Emphasis Type="Italic">Diplazium</Emphasis> hybrids involving <Emphasis Type="Italic">D. plantaginifolium</Emphasis> and <Emphasis Type="Italic">D</Emphasis>. <Emphasis Type="Italic">ternatum</Emphasis> from Mexico and Central America

Here we evaluate the origins and relationships of Mexican and Central American Diplazium hybrids derived from crosses involving either D. plantaginifolium or D. ternatum. Based on study of live plants and herbarium specimens, we distinguish D. ×verapax from the similar D. riedelianum and present evidence that the former is a sterile hybrid derived from a cross between D. plantaginifolium and D. werckleanum. We also describe new hybrids, D. ×torresianum and D. ×subternatum from Mexico and northern Central America. Both involve D. ternatum as one parent. Diplazium. cristatum is the other putative parent of D. ×torresianum, and D. plantaginifolium is the second parent of D. ×subternatum. We also designate lectotypes for D. cordovense and D. dissimile.     

<Emphasis Type="Italic">Candida krusei</Emphasis> and <Emphasis Type="Italic">Candida glabrata</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> by downregulating expression of <Emphasis Type="Italic">HWP1</Emphasis> gene

Pathogenicity of Candida albicans is associated with its capacity switch from yeast-like to hyphal growth. The hyphal form is capable to penetrate the epithelial surfaces and to damage the host tissues. Therefore, many investigations have focused on mechanisms that control the morphological transitions of C. albicans. Recently, certain studies have showed that non-albicans Candida species can reduce the capacity of C. albicans to form biofilms and to develop candidiasis in animal models. Then, the objective of this study was to evaluate the effects of Candida krusei and Candida glabrata on the morphogenesis of C. albicans. Firstly, the capacity of reference and clinical strains of C. albicans in forming hyphae was tested in vitro. After that, the expression of HWP1 (hyphal wall protein 1) gene was determined by quantitative real-time PCR (polymerase chain reaction) assay. For both reference and clinical strains, a significant inhibition of the hyphae formation was observed when C. albicans was incubated in the presence of C. krusei or C. glabrata compared to the control group composed only by C. albicans. In addition, the culture mixed of C. albicans-C. krusei or C. albicans-C. glabrata reduced significantly the expression of HWP1 gene of C. albicans in relation to single cultures of this specie. In both filamentation and gene expression assays, C. krusei showed the higher inhibitory activity on the morphogenesis of C. albicans compared to C. glabrata. C. krusei and C. glabrata are capable to reduce the filamentation of C. albicans and consequently decrease the expression of the HWP1 gene.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

The <Emphasis Type="Italic">Adh</Emphasis> locus and adaptation of <Emphasis Type="Italic">cn</Emphasis> and <Emphasis Type="Italic">vg</Emphasis> mutants in experimental populations of <Emphasis Type="Italic">Drosophila melanogaster MEIG</Emphasis>

A complex study on the adaptation of cn and vn mutants and the allozymes of alcoholdehydrogenase (ADH) was carried out in initially pure lines, and their panmixia populations during exchange of the mutant genotype with that of wild-type flies (C-S) and D) through saturating crossings. The relative adaptation of the genotypes was estimated by their effect on reproductive efficiency in the experimentally obtained population. Fecundity, lifespan, and the resistance of the studied genotypes to hyperthermia were investigated individually. It was shown that the high level of adaptation of the cn mutants and the low level of adaptation of the vg mutants was correlated with the presence of different ADH allozymes. In the studied population, the F-allozyme of ADH accompanied the vg mutation, while the S-allozyme of the enzyme was detected in cn mutants. Saturating crossings of C-S(Adh ^S)×vg(Adh ^F) and D(Adh ^F) × cn(Adh ^S), along with the parallel determination of the allele composition of the Adh locus, demonstrated that the complete substitution of the F-allozyme of ADH in the vg mutants by the S-allozyme in D flies, as well as the substitution of the S-allozyme of ADH in the cn mutants by the F-allozyme in D flies was realized only after the 15th–20th backcrosses. These results favor the coadaptation of cn and vg marker genes with alleles of the Adh locus and indicate the important role of the latter in the adaptation of genotypes. In the studied population, selection acted primarily against the vg mutants, which were inferior to the cn mutants, and heterozygote genotypes in indices of the main adaptation components.     

Genome-wide identification and evolution of <Emphasis Type="Italic">TC1</Emphasis>/<Emphasis Type="Italic">Mariner</Emphasis> in the silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis>) genome

TC1/Mariner transposons belong to class II transposable elements (TEs) that use DNA-mediated “cut and paste” mechanism to transpose, and they have been identified in almost all organisms. Although silkworm (Bombyx mori) has a large amount of TC1/Mariner elements, the genome wide information of this superfamily in the silkworm is unknown. In this study, we have identified 2670 TC1/Mariner (Bmmar) elements in the silkworm genome. All the TEs were classified into 22 families by means of fgclust, a tool of repetitive sequence classification, seven of which was first reported in this study. Phylogenetic and structure analyses based on the catalytic domain (DDxD/E) of transposase sequences indicated that all members of TC1/Mariner were grouped into five subgroups: Mariner, Tc1, maT, DD40D and DD41D/E. Of these five subgroups, maT rather than Mariner possessed most members of TC1/Mariner (51.23%) in the silkworm genome. In particular, phylogenetic analysis and structure analysis revealed that Bmmar15 (DD40D) formed a new basal subgroup of TC1/Mariner element in insects, which was referred to as bmori. Furthermore, we concluded that DD40D appeared to intermediate between mariner and Tc1. Finally, we estimated the insertion time for each copy of TC1/Mariner in the silkworm and found that most of members were dramatically amplified during a period from 0 to 1 mya. Moreover, the detailed functional data analysis showed that Bmmar1, Bmmar6 and Bmmar9 had EST evidence and intact transposases. These implied that TC1/Mariner might have potential transpositional activity. In conclusion, this study provides some new insights into the landscape, origin and evolution of TC1/Mariner in the insect genomes.     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Fungicidal activity of cellobiose lipids from culture broth of yeast <Emphasis Type="Italic">Cryptococcus humicola</Emphasis> and <Emphasis Type="Italic">Pseudozyma fusiformata</Emphasis>

Cellobiose lipids of yeast fungi Cryptococcus huminola and Pseudozyma fusiformata have similar fungicidal activities against different yeast, including pathogenic Cryptococcus and Candida species. Basidiomycetic yeast reveals maximum sensitivity to these preparations; e.g., cells of cryptococcus Filobasidiella neoformans almost completely die after 30-min incubation in a glycolipid solution at a concentration of 0.02 mg/ml. The same effect toward ascomycetous yeast, including pathogenic Candida species, is achieved only at five to eight times higher concentrations of glycolipids. The cellobiose lipid from P. fusiformata, which, unlike glycolipid from Cr. humicola, has hydroxycaproic acid residue as O-subtituent of cellobiose and additional 15-hydroxy group in aglycone, inhibits the growth of the studied mycelial fungi more efficiently than the cellobiose lipid from Cr. humicola.     

Expression of gene for <Emphasis Type="Italic">N</Emphasis>-acyl-homoserine lactonase <Emphasis Type="Italic">AiiA</Emphasis> affects properties of rhizospheric strain <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

The introduction into strain Pseudomonas chlororaphis 449 of plasmid pME6863 that contains the cloned gene for N-acyl-homoserine lactonase, AiiA, leads to the degradation of all three types of N-acylhomoserine lactones produced by this strain (N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, and N-3-oxo-hexanoyl-homoserine lactone). This causes a drastic reduction in the synthesis of phenazine pigment and decreases the ability of cells to migrate on the surface of nutrient medium. However, the antagonistic activity of P. chlororaphis 449 toward phytopathogenic fungi Sclerotinia sclerotiorum and Rhizoctonia solani is not only decreased, but is even slightly increased; no essential changes in the exoprotease activity were observed. It is assumed that one of the QS systems of P. chlororaphis 449 may exert the repression effect on the expression of genes, which determine the two latter cell activities.